We demonstrate a robust high power 930 nm femtosecond Nd:fiber laser system with hollow-core photonic bandgap fiber (HC-PBGF) as the output delivery, which can be easily integrated into compact two-photon microscopy system for bio-imaging. The whole laser system can deliver up to 17.4 nJ, 220-fs pulses at 930 nm with repetition rate of 46 MHz. In this paper, this laser was demonstrated as the light source for volumetric imaging of zebrafish blood vessel.
© 2017 Optical Society of America
Since the first demonstration in 1990 , two-photon microscopy (TPM) has undergone tremendous development and been applied to cell biology and the neuron sciences. Inasmuch as the solid-state femtosecond lasers are developed contemporarily with TPMs, they dominate the applications of TPM. Still, femtosecond fiber lasers have attracted much attention in recent years and been considered as an ideal light source for TPM, for they show such advantages as the compactness, robustness and simplicity of operation over conventional solid-state femtosecond lasers, such as Ti:sapphire lasers .
Recently, various compact multiphoton microscopes have been reported using mode-locked fiber laser [3,4]. The wavelengths are mainly focused on 1030 nm, using well-developed Yb-doped fibers. While the TPM at the 920 nm spectral window enables two-photon excitation of a variety of green fluorophores, such as the current generations of protein-based genetically encoded calcium indicators (GCaMP,e.g.) . Therefore, the development of ~900 nm femtosecond fiber lasers has great potential applications as laser sources for TPM.
Compared with the femtosecond pulses produced by nonlinear wavelength conversion in a high-nonlinearity fiber [6,7], the direct generation of 900-nm-range laser in Nd-doped fiber is preferred due to its robustness, simplicity and high stability. However, either the laser oscillation or the amplification at that wavelength has to compete with four-level transition (4F3∕2 − 4I11∕2) at 1060 nm [8,9]. We have demonstrated the direct generation of femtosecond pulses at 920 nm in a core-pumped Nd:fiber laser . By pre-chirped amplification, we also achieved 121 mW average power and 4.4 nJ pulse energy  and obtained the in vivo imaging of a zebrafish with that laser.
Furthermore, newly-developing volumetric TPM [12,13] enhances the energy requirements for femtosecond laser pulses. Differ from standard TPM, in which the excitation region is limited to the focal point, volumetric TPM uses axially elongated Bessel beam to excite the sample simultaneously. Higher pulse energy is always preferred. However, in our previous core-pumped Nd fiber laser system [10,11], the pump power of single-mode 808 nm laser diode (<250 mW) limits the final output power. The combination of W-type double-cladding Nd-doped fiber and high power multimode 808nm pump diode shows the potential to scale the power at ~900 nm , which is used in our new fiber amplifier design.
Delivery of the laser pulses from the laser to the microscope is another important issue. Rather than the direct free-space coupling, fiber-based delivery is preferred due to the benefits of vibration isolation and flexible TPM system design. With low dispersion and nonlinearity, hollow-core photonic bandgap fiber (HC-PBGF) is regarded as an ideal medium for high power femtosecond pulses delivery without pulse deterioration [15,16]. However, the transmission band of the PBGF needs to be carefully designed, determined by the fiber cladding structure. Without the commercial hollow-core fiber at this band, we designed and fabricated this specific 900 nm band HC-PBGF for our TPM.
In this paper, we demonstrate the 930 nm fiber laser with the pulse energy up to 17.4 nJ and the total output power of 800 mW after the pulse compressor. The pulse duration is measured to be 220 fs pulses with the repetition rate of 46 MHz. After fiber coupling and propagation, the output from the hollow-core fiber was up to 550 mW with the coupling efficiency of 69%. By this high power fiber-pigtailed femtosecond pulses, we can easily image the blood vessel of a zebrafish in volumetric TPM based on Bessel beam.
2. Experimental setup
The experiment setup of the fiber laser is schematically illustrated in Fig. 1. The system consists of a mode-locked ring Nd:fiber oscillator and a double-cladding amplifier.
A compact and intracavity dispersion compensated Nd:fiber laser serves as the seed source. The single mode Nd-doped fiber was used as the gain fiber, which has higher doping concentration of neodymium, compared with the following W-type fiber in the amplifier stage. The cavity configuration includes a gain fiber, two wavelength division multiplexers (WDMs), two collimators, a grating pair and several free-space polarization controlling components. The gain fiber is about 3.5 m long with the absorption of ~8 dB/m at 808 nm. Two polarization beam splitters (PBSs), a Faraday rotator and a half-wave plate serve as an intracavity isolator to ensuring the one directional operation of the laser. The grating pair is a transmission type in the groove density of 1850 lines/mm. The slant grating distance is set to be 2.5 mm. Two pump lasers with the total power of 400 mW are coupled from both sides of the gain fiber through two WDMs into the cavity. The output pulses are rejected from the PBS. Compared with the all-normal dispersion regime we reported in Ref. 7, the dispersion compensated regime requires lower pumping threshold, which is about 200 mW. The oscillator can be mode-locked both at 910 nm and 930 nm by tuning the collimators in the cavity. Given that the amplified spontaneous emission (ASE) of gain fiber in the amplifier centered at 930 nm, the oscillator was tuned the same to obtain a higher match, thereby a higher amplification efficiency. The key difference from our previous version is that, unlike the commonly used σ cavity for roundtrip grating pass, we employed a transmission type grating pair in single-pass configuration, shown in Fig. 1(a). The main reason is that the filtering effect caused by the single-pass grating configuration can help to block the four-level transition at 1060 nm, shown in Fig. 1(b). Also, the single-pass to the grating pair can reduce the transmission loss compared with the double-pass configuration.
The main difficulty in the amplification of the pulses at 930 nm using Nd-doped fiber is the competition with the transition at 1060 nm. We chose to use the W-type Nd-doped fiber in the amplifier . The W-type fiber is a double-cladding fiber whose refractive index in the inner cladding is less than in the outer cladding. This kind of fiber can provide the cutoff wavelength, for the fundamental LP01 mode. Light at the wavelength longer than the cutoff will leak off this fiber. Besides, the cutoff wavelength can also be shifted by bending the fiber in different radii of curvature which can also add further loss to the unwanted long wavelength emissions. In this experiment, the W-fiber has the LP01 cutoff wavelength at about 1 μm. Then the emission at 1060 nm during the amplification can be effectively suppressed, while high transparency at 930 nm range can also be maintained. By this way, the signal is effectively amplified in the fiber.
Figure 1(c) depicts the amplifier configuration. In order to avoid the nonlinear effect such as the self-phase modulation and Raman shift in the high power amplification, the conventional chirped pulse amplification  was applied. After passing through another isolator, the pulses are stretched to about 35 ps by a 30 m long SMF, and then injected into the fiber amplifier. The W-type Nd:fiber is 9 m long with the radius of curvature adjusted to 14.5 cm and was pumped by a 12 W multi-mode fiber coupled diode laser. At the output end, the gain fiber was fusion spliced with a single-mode collimator. The amplified pulses were launched out through the collimator, while the unabsorbed pump laser in the inner cladding leaked out through the splice.
After the amplification, the pulses were de-chirped by a grating pair with a groove density of 1500 lines/mm, and they were coupled into a 1 m-long HC-PBGF with the efficiency of about 69%. The HC-PBGF end face was collapsed to avoid contamination by the moist air. Given that the HC-PBGF would introduce small negative chirp to the pulses, the compressor can be adjusted to keep the pulses to its shortest at the output.
3. Nd-doped fiber laser performance
The spectra of the seed and the amplified pulses are shown in Fig. 2(a). The spectral bandwidths of the respective pulses are 20 nm and 11 nm. The mismatch was due to the different kinds of gain fiber used in oscillator and amplifier. Figure 2(b) shows both the emission at 930 nm and 1060 nm of the W-type fiber, in which the emission at 1060 nm is lower than 930 nm by a factor of 26 dB.
The inset in Fig. 2(b) also illustrates the cross section of the W-type fiber and the corresponding cut-off wavelength. The output pulses of 50 mW pulses with repetition rate of about 46 MHz were injected into the amplifier, then amplified to a maximum of 1.1 W at a pump power of 12 W and the slope efficiency is about 10.8%, shown in Fig. 2(c). When the radius of W-type Nd:fiber curvature was shorter than 14.5 cm, the amplification efficiency dropped. While the radius is larger, the emission at 1060 nm turns out to present, which would cause low amplification efficiency of signal at 930 nm as well. Due to the high groove density and the limited size of the grating, the emission at 1060 nm was separated with 930 nm and filtered out .The pulses were compressed to its shortest, with compress efficiency of about 70%. The calculated and measured intensity autocorrelation traces are superimposed in Fig. 2(d). The pulse duration was measured to be 220 fs, corresponding to 1.22 times of Fourier transform limited pulses.
For the delivery, the amplified pulse was coupled into a 1-m-long HC-PBGF. The fiber core is about 8 μm in diameter and surrounded with a ~152 μm diameter cladding layer. The transmission loss of this fiber is about 101 dB/km at 930 nm. After the HC-PBGF, the pulses were stretched to 530 fs, and then re-compressed to 220 fs by the same compressor in the amplifier. The intensity autocorrelation traces are shown in Fig. 3(a). This indicates that the HC-PBGF does not introduce the additional nonlinearity to the pulses. Figure 3(b) presents the beam profile after the HC-PBGF collimator. The optical attenuation of the fiber and the group-velocity dispersion (GVD) are shown in Fig. 3(c).
4. Volumetric two-photon imaging test
To test the system performance, we conducted an imaging test in our homemade volumetric two-photon microscopic system to demonstrate its high power application. The schematic of the system is illustrated in Fig. 4. The laser pulses were launched into the microscope through the hollow-core fiber. The collimator contains a beam expander to expand the diameter of the laser to 12 mm. We use a refractive axicon with the angle α of 2°to generate a Bessel beam. By scanning the needle-like Bessel beam, we could obtain the image of a 500 × 500 × 250 μm3 volume with a single scan at 1 Hz. The more detailed devices information has been given in reference .
Figure 5 shows the in vivo imaging of the blood vessel of a zebrafish (2 days post-fertilization). The zebrafish is the Tg (kdrl:EGFP) transgenic fish line, with the vascular epithelial cells labeled with EGFP. The fish was raised in embryo medium containing 0.002% phenylthiourea (PTU, Sigma). Prior to live imaging, the zebrafish was anaesthetized with 0.01% tricaine (Sigma), and embedded in a 1% ultra-pure agarose (Invitrogen). The postobjective power of about 110 mW was used, with the microscopic system efficiency of about 25%.
We demonstrated a W-type Nd-doped fiber amplifier operating at 930 nm, which offers an average power of 800 mW 220-fs pulses at 46 MHz, corresponding to a single pulse energy of 17.4 nJ. The fiber delivered power is about 550 mW. The volumetric imaging of zebrafish blood vessel verified the performance of this whole system and showed the great potential of this laser as a new light source for two-photon fluorescence imaging and other applications.
This work was supported by the National Natural Science Foundation of China (Grant 61475008, 31327901).
The authors are grateful to Meijun Pang, Zhuan Zhou, and Heping Cheng from the Institute of Molecular Medicine, Peking University for their assistance in the TPM imaging experiments.
References and links
3. G. Liu, K. Kieu, F. W. Wise, and Z. Chen, “Multiphoton microscopy system with a compact fiber-based femtosecond-pulse laser and handheld probe,” J. Biophotonics 4(1-2), 34–39 (2011). [CrossRef] [PubMed]
6. W. Liu, C. Li, Z. Zhang, F. X. Kärtner and G. Chang, “Yb-fiber laser based ultrafast source emitting 50-fs pulses at 920 nm,” in Ultafast Optics 2015, paper 0123.
7. J. R. Unruh, E. S. Price, R. G. Molla, L. Stehno-Bittel, C. K. Johnson, and R. Hui, “Two-photon microscopy with wavelength switchable fiber laser excitation,” Opt. Express 14(21), 9825–9831 (2006). [CrossRef] [PubMed]
8. K. Arai, H. Namikawa, and T. Handa, “Aluminum or phosphorus co-doping effects on the fluorescence and structural properties of neodymium doped silica glass,” J. Appl. Phys. 59(10), 3430–3436 (1986). [CrossRef]
9. P. D. Dragic and G. C. Papen, “Efficient Amplification Using the 4F3/2 − 4I9/2 Transition in Nd-Doped Silica Fiber,” IEEE Photonics Technol. Lett. 11(12), 1593–1595 (1996). [CrossRef]
10. X. Gao, W. Zong, B. Chen, J. Zhang, C. Li, Y. Liu, A. Wang, Y. Song, and Z. Zhang, “Core-pumped femtosecond Nd:fiber laser at 910 and 935 nm,” Opt. Lett. 39(15), 4404–4407 (2014). [CrossRef] [PubMed]
11. B. Chen, T. Jiang, W. Zong, L. Chen, Z. Zhang, and A. Wang, “910nm femtosecond Nd-doped fiber laser for in vivo two-photon microscopic imaging,” Opt. Express 24(15), 16544–16549 (2016). [CrossRef] [PubMed]
12. R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017). [CrossRef] [PubMed]
14. M. Laroche, B. Cadier, H. Gilles, S. Girard, L. Lablonde, and T. Robin, “20 W continuous-wave cladding-pumped Nd-doped fiber laser at 910 nm,” Opt. Lett. 38(16), 3065–3067 (2013). [CrossRef] [PubMed]
15. S. P. Tai, M. C. Chan, T. H. Tsai, S. H. Guol, L. J. Chen, and C. K. Sun, “Two-photon fluorescence microscope with a hollow-core photonic crystal fiber,” Opt. Express 12(25), 6122–6128 (2004). [CrossRef] [PubMed]
16. W. Zong, R. Wu, M. Li, Y. Hu, Y. Li, J. Li, H. Rong, H. Wu, Y. Xu, Y. Lu, H. Jia, M. Fan, Z. Zhou, Y. Zhang, A. Wang, L. Chen, and H. Cheng, “Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice,” Nat. Methods 14(7), 713–719 (2017). [CrossRef] [PubMed]
17. S. Yoo, D. B. S. Soh, J. Kim, Y. Jung, J. Nilsson, J. K. Sahu, J. W. Lee, and K. Oh, “Analysis of W-type waveguide for Nd-doped fiber laser operating near 940 nm,” Opt. Commun. 247(1-3), 153–162 (2005). [CrossRef]
18. P. Maine, D. Strickland, P. Bado, M. Pessot, and G. Mourou, “Generation of ultrahigh peak power pulses by chirped pulse amplification,” IEEE J. Quantum Electron. 24(2), 398–403 (1988). [CrossRef]